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284:429-438, 2003. doi:10.1152/ajpcell.00261.2002Am J Physiol Cell PhysiolNadia A. Ameen, Christopher Marino and Pedro J. I. SalasCFTR and fluid transport in rat jejunum in vivocAMP-dependent exocytosis and vesicle traffic regulate

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CHE cells are also present in the human small intes-tine suggests that cAMP-dependent exocytosis ofCFTR is also a potentially important regulatory mech-anism in the human intestine (3, 34).

The extent to which vesicle traffic regulates fluidsecretion and the number of CFTR channels expressedon the surface of the small intestinal lumen (the pre-dominant site of CFTR-mediated fluid secretion) is

unknown. In previous studies (2), we used immuno-electron microscopy to examine and quantify the sub-cellular distribution of CFTR. These observations con-firmed that CFTR was associated with subapicalvesicles and the plasma membrane of both crypt andCHE cells. Furthermore, quantification of the subcel-lular distribution in these cells supported a role forCFTR regulation by vesicle traffic in the rat smallintestine (2). On the basis of these observations, weused independent morphological and biochemicalmethodologies in conjunction with fluid secretion mea-surements in the current study to determine whethercAMP and vesicle traffic regulate the exocytosis ofCFTR to the apical membrane of the crypts and thewhole mucosa of rat proximal small intestine.

MATERIALS AND METHODS

Animal preparation and fluid secretion measurements. Thestudy was approved by the Animal Research Committee ofthe University of Miami School of Medicine. Male Sprague-Dawley rats (Charles River Laboratories) weighing 250300g were fed standard chow. Rats were fasted overnight andanesthetized with 45 mg/kg pentobarbital sodium adminis-tered intraperitoneally. After anesthesia, a tracheotomy wasperformed to maintain a patent airway. Rectal temperatureof 38.1C was maintained with a thermostatically controlledheating lamp. Rats were subjected to a laparotomy via a

midline incision, a 30-cm length of jejunum was identifiedand cannulated proximally and distally, and the lumen waswashed with warm 0.9% NaCl. The saline was removed fromthe loop by blowing through the proximal cannula. The distalcannula was removed, and ligatures were placed to createfour intestinal loops each 4 cm in length with a 2-cm lengthof intestine separating each loop. Equal volumes (0.5 ml) ofdrug [0.1 mM primaquine 1.0 mM 8-bromo-cAMP (8-BrcAMP), 10 g/ml nocodazole 1.0 mM 8-BrcAMP, 1.0 mM8-BrcAMP, or PBS] were delivered into each loop by a fluid-filled syringe that was weighed before (A, g) and after (B, g)delivery into each loop. The abdomen was closed, and theanimal was observed for 2 h. At the end of the study period,the abdomen was opened and intestinal loops were excised,blotted free of excess fluid, and weighed (C, g). The loops were

then cut open, drained, blotted, and reweighed (D, g). Theamount offluid recovered was obtained by subtraction (C

D, g). The net movement of fluid into or from the loop wascalculated as [(C D) (A B)] (g). Fluid absorption wasreflected by a net loss offluid from the loop, (A B) (C

D), and secretion occurred if there was a net gain offluid intothe loop, (C D) (A B). Because the densities of thesolutions weighed are all approximately equal to that ofwater, the fluid weights are assumed to be identical withfluid volumes. Fluid movements in or out of the loops werecalculated as micrograms per centimeter of jejunal length perminute. At the end of the study period, intestinal tissuesfrom each loop were embedded in OCT embedding medium,

frozen in liquid nitrogen-cooled isopentane, and prepared forimmunocytochemistry as described previously (3, 4).

Independent studies were performed to confirm our observations of CFTR distribution in the intestine after cAMPstimulation with the receptor agonist vasoactive intestinalpeptide (VIP) as described previously (4). Thirty minutesafter VIP administration tissues from rat jejunum were re-moved, embedded in OCT embedding medium, frozen, andprepared for indirect immunofluorescence labeling, and en-

terocytes were isolated and used in immunoprecipitation andsurface biotinylation studies.

Reagents. All chemicals were obtained from Sigma (St.Louis, MO) except where stated. R3194 and R3195 are affinity-purified polyclonal antibodies raised against rodent CFTRand were provided by C. R. Marino. The specificity of bothantibodies has been documented in rats (1, 2, 4, 44). Thepreviously characterized antibody to lactase, YBB 2/61, was agift from Dr. A. Quaroni (Cornell University, Ithaca, NY; Ref.29). The antibody to AKAP149 was purchased from AlomoneLaboratories (Jerusalem, Israel).

Isolation of intestinal enterocytes. Segments of rat jejunumwere removed 30 min after VIP administration, and villusand crypt enterocytes were isolated as described previously(24). Suspensions of freshly isolated enterocytes were subject

to Trypan blue exclusion, immunofluorescence immunocyto-chemistry, and surface biotinylation studies.

Surface biotinylation of isolated enterocytes. Suspensionsof freshly isolated cells from rat small intestine after VIP ordiluent infusion were biotinylated by rotating in the cold for30 min in freshly prepared 1.0 mM sulpho-NHS biotin [bicin-choninic acid (BCA) protein assay kit; Pierce Laboratories,Rockford, IL] in PBS-CM (PBS supplemented with 1.3mMCa2Cl and 1.0 mM Mg2Cl). Control cells were incubated withPBS-CM. After biotinylation, cells were washed in the coldwith 50 mM NH4Cl in PBS-CM to quench unreacted freebiotin and immunoprecipitation was performed. Surface bi-otinylation of CFTR from the lumen of the intact intestinewas also attempted; however, complete diffusion into thedeep crypts (a major site of CFTR expression) could not be

ensured. Surface biotinylation was therefore performed inisolated cells because the yield of isolated crypt and villusenterocytes was high in our hands and polarity and mem-brane preservation could be ensured in the majority of cells.Indeed, it has been shown that isolated enterocytes remainwell polarized for hours (45), a fact that we tested further inour experiments (see Fig. 3E).

Immunoprecipitation of CFTR. Enterocytes were lysed inimmunoprecipiation buffer (IP) containing 0.5% Triton

X-100, 0.1% SDS, and 0.5% sodium deoxycholate in PBS pH7.4 supplemented with a protease inhibitor cocktail (Sigma).Samples were homogenized and sonicated and then centri-fuged for 15 min in the cold at 14,000 rpm. Protein contentwas measured by UV absorption (Pierce Laboratories), andimmunoprecipitation was performed with a minimum of 1

mg/ml protein in supernatants. Supernatants were pre-cleared with protein A-Sepharose beads (Amersham Phar-macia, Piscataway, NJ) for 1 h in the cold. Immunoprecipi-tations were performed with the anti-CFTR antibody R3194,antibody to lactase YBB2/61, or nonspecific rabbit IgG. An-tibody or serum was added to supernatants and incubated for2 h at 4C. Protein A agarose (5 mg/sample) was added tosamples and resuspended in IP buffer containing 1% (wt/vol)globulin-free bovine serum albumin (BSA), and then sampleswere rotated overnight in the cold. Samples were then cen-trifuged (14,000 rpm), supernatants were discarded, and thebeads were washed in IP buffer supplemented with 0.5 MKCl. The immunoprecipitates were eluted in 1% SDS, 4 M

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urea, and 1 mM Tris HCl, pH 6.8, precipitated on ice intrichloroacetic acid, acetone extracted, and air dried. Thedried pellets were resuspended in sample buffer before anal-ysis by Western blot.

Western blotting. Immunoprecipitates were analyzed bySDS-PAGE using a 7.5% gel and proteins transferred topolyvinylidene difluoride (PVDF) membranes by a semi-drytransfer method. After transfer of proteins, membranes werewashed in deionized water, nonspecific proteins were blocked

in PBS-0.05% Tween 20 containing 5% nonfat dry milk for2 h, and biotinylated proteins were detected by streptavidinperoxidase binding (Sigma). Western blots of nonbiotinylatedimmunoprecipitates were analyzed with antibodies to CFTR(R3194) and lactase (YBB2/61). Western blots of CFTR or IgGimmunoprecipitates were also analyzed with a commercialantibody to AKAP 149 (Alomone Labs). Detection of primaryantibodies was accomplished by using goat anti-rabbit (1:10,000) or anti-mouse (1:8,000) peroxidase secondary anti-bodies (Sigma). After immunodetection, membranes wereexposed to chemiluminescence (preflashed Hyperfilm ECL,

Amersham Pharmacia). Densitometric analysis of proteinbands was performed with a Kodak Image Station 440 CFand IS440CF image analysis software. Bands were selected,and signal was weighted as number of pixels (average pixel

intensity in the band average pixel intensity in the back-ground).

Immunocytochemistry. Intestinal tissues from all experi-ments were prepared for immunocytochemical localizationstudies with indirect immunofluorescent immunolabeling ofcryostat sections from jejunum as described previously (1, 4),and labeled sections were examined on a Leica epifluorescentmicroscope. Confocal microscopy and image analysis of CFTRfluorescence intensities were performed as described previ-ously (4) with a Zeiss LSM 510 microscope equipped withimage analysis software. The apical domain of CFTR inlabeled sections was determined from images of perpendicu-lar parallel sections labeled with fluorescent phalloidin andmeasured 1.5 m in length from the luminal surface. TheCFTR signal below that depth was considered the subapical

compartment. Acquisition of parameters were adjusted withthe software so that the pixel intensity of the brightestfluorescence was not saturated (255 pixels). Data was col-lected from an average of 30 cells in random sections (aver-age 10 sections) from each tissue examined.

Indirect immunofluorescence labeling for apical mem-brane markers (lactase and CFTR) was performed on freshlyisolated enterocytes from rat jejunum to confirm preserva-tion of polarity. A drop of cell suspension was placed onto apoly L-lysine-coated slide and allowed to air dry. Briefly, cellswere fixed in 2% paraformaldehyde for 10 min and washed in50 mM ammonium chloride, and nonspecific proteins wereblocked in PBS-BSA 1% for 30 min. Cells were exposed toprimary antibody or PBS-BSA 1% for 1 h at room tempera-ture in a moist chamber. Primary antibody was detected with

FITC-conjugated secondary antibody diluted in PBS-BSA1%. After immunolabeling, slides were examined with aLeica epifluorescent microscope.

RESULTS

Morphological distribution of CFTR in rat jejunumafter cAMP stimulation. Our previous light microscopiclocalization of CFTR in rat proximal small intestinalcrypts revealed a subapical distribution for CFTR, sug-gesting the presence of CFTR in a vesicular compart-ment. Immunoelectron microscopic examination re-vealed that although CFTR was detected on the apical

membrane, the majority of CFTR was associated withsubapical vesicles, supporting a role for vesicle inser-tion in regulating CFTR and anion secretion in thecrypt (2). VIP, a cAMP agonist, also induced a redistri-bution of CFTR from the subapical compartment to theapical domain in villus CHE cells as observed previ-ously (4). On the basis of these observations, we exam-ined the distribution of CFTR in the crypt after VIP

(Fig. 1E). We compared the distribution with that oflactase, an integral apical membrane protein that isnot regulated by cAMP-dependent vesicle traffic (Fig.1, A and B; Refs. 23, 26). Although lactase is absent inproliferative undifferentiated crypt cells and the high-est levels of CFTR are found in this compartment, bothlactase and CFTR are present on the apical pole ofnewly differentiated crypt cells that are more superfi-cially located (Fig. 1, A and D; Refs. 3, 29, 34). In fact,lactase is mostly found in a subapical compartment inthe crypts (Fig. 1, A and B), whereas it is expressed inthe brush border in the villus (29). Examination ofcrypt sections from rat jejunum revealed that lactasedid not redistribute to the cell surface after VIP admin-

istration (Fig. 1, B compared with A). CFTR, on theother hand, was distributed in a broad subapical regionunder control conditions (Fig. 1D) and redistributed tothe apical surface in a narrow band (correlating withthe region of phalloidin label) after VIP (Fig. 1E). Tofurther confirm that the effect of VIP was mediated bycAMP, similar experiments were conducted after lumi-nal 8-BrcAMP stimulation. Examination of labeled sec-tions by confocal microscopy after administration of themembrane-permeant agonist 8-BrcAMP similarly con-firmed a redistribution of CFTR from a predominantsubapical compartment (Fig. 2A) to the region corre-sponding to the apical microvilli of crypt epithelial cells

(Fig. 2B). The 8-BrcAMP-dependent shift of CFTRfrom the subapical to apical domain corresponded withan almost threefold increase in the ratio of apical tosubapical CFTR fluorescence (6.15 3.08) comparedwith unstimulated PBS controls (2.14 1.03; P 0.001) and was associated with net fluid movementinto the lumen of the jejunum (Table 1).

To determine whether the cAMP-dependent shift ofCFTR from the subapical compartment to the apicaldomain of crypt cells was dependent on vesicle traffic,we examined whether the same shift of CFTR signaloccurred when vesicle traffic was interrupted. The an-timalarial drug primaquine is a lysosomotropic aminethat inhibits vesicle trafficking and prevents the fusion

of vesicles with the plasma membrane (16). It wasrecently shown to be a potent inhibitor of CFTR vesicletraffic in oocytes (39). Examination of crypt sectionsfrom jejunum labeled for CFTR after pretreatmentwith 8-BrcAMP and primaquine (Fig. 2C) revealed aprominent rim of CFTR fluorescence label (extending1.5 m) beneath the apical microvilli, similar to thesubapical distribution of CFTR under control condi-tions (Fig. 1A). The CFTR apical fluorescence wasreduced in the presence of primaquine both in terms ofpixel values and in the apical-to-subapical ratio (Table1). Accordingly, the reduction in the shift of CFTR from

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the subapical to the apical domain after pretreatmentwith primaquine and 8-BrcAMP was associated withan increase in CFTR signal detected in the subapicalcompartment in the presence of primaquine. The shiftin the subcellular distribution of CFTR in the cryptparalleled a functional effect of primaquine pretreat-ment in blocking the fluid secretory response of8-BrcAMP by 35% (Table 1). These observations areconsistent with an effect of primaquine in inhibitingthe 8-BrcAMP-dependent insertion of CFTR-contain-ing vesicles from the subapical compartment into theapical membrane and suggest that cAMP and vesicle

insertion of CFTR to the apical membrane regulatesfluid secretion in the jejunum.

Microtubules serve as molecular motors in the trans-port of vesicles from the Golgi complex to the apicaldomain of polarized cells and have been shown to playa role in cAMP-dependent exocytosis of CFTR in T84cells and in cAMP-dependent chloride secretion in ratcolon (14, 10, 21, 38). However, studies of polarizedMadin-Darby canine kidney (MDCK) cells and airwayepithelial cells could not confirm a role for microtu-bules in regulating the exocytosis of CFTR (22, 25). Weexamined the distribution of CFTR after 8-BrcAMP

Fig. 1. Vasoactive intestinal peptide (VIP) induces a redistribution of the cystic fibrosis transmembrane conduc-tance regulator (CFTR), but not lactase, to the apical membrane of crypt cells. Lactase fluorescence labeling incrypt cells under control conditions (A) reveals a distribution under the apical domain that does not change after

VIP stimulation (B). Control section labeled with nonimmune serum reveals lack of specific staining (C). Underunstimulated conditions, CFTR fluorescence (D) is distributed in a broad band in the subapical region (arrows) andredistributes to the apical surface after VIP stimulation (E). Control section labeled with CFTR antibody (R3195)preincubated with peptide (F). Bars, 10 m.



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stimulation in the presence of nocodazole, an agentthat blocks microtubule polymerization and therebyprevents vesicle transport in cells. Nocodazole alsoblocked the subapical-to-apical shift in CFTR signal,although to a lesser extent than primaquine, and re-duced the 8-BrcAMP-induced fluid secretory responseby 33% (Table 1). Because the mechanisms of action ofprimaquine and nocodazole are different and they havein common their effect on exocytic membrane traffic,these results strongly suggest a role of cAMP-inducedexocytosis of CFTR as a mechanism to regulate CFTR-mediated anion transport and fluid secretion.

Detection of CFTR exocytosis by surface biotinylationin vivo. Although immunofluorescence examination ofthe distribution of CFTR in intestinal sections aftercAMP agonist treatment suggested a shift of CFTRfrom a subapical compartment to the apical domain, wewished to independently confirm that cAMP indeedstimulated exocytosis of CFTR to the surface of intes-tinal cells. Immunolocalization in toto could not con-firm this because our antibodies were raised againstthe cytoplasmic COOH terminus of CFTR. We there-fore used the technique of surface biotinylation, a sen-

sitive method that is widely used to quantify surfaceproteins in cells and to study membrane traffic ofproteins and has been used in studies examining CFTRmembrane traffic (28, 30).

Morphological examination of fixed intestinal seg-ments after isolation confirmed that we could success-fully retrieve most cells for biotinylation and immuno-precipitation, including those from the crypts, within30 min. Lack of damage to isolated enterocytes wasconfirmed by Trypan blue exclusion in cell suspen-sions. Furthermore, immunofluorescence labeling offreshly isolated cells confirmed preservation of polarityby the presence of apical markers (Fig. 3, E and F) inisolated cells. Immunoprecipitations were performedon freshly isolated cells with the CFTR antibody R3194and nonspecific rabbit IgG as negative controls, andimmunoprecipitates were analyzed for CFTR by West-ern blots using the same CFTR antibody. Western blotanalysis of immunoprecipitates from VIP-stimulatedor control cells with R3194 detected a broad band ofmature CFTR (band C) of molecular mass of 170185kDa (Fig. 3A, lane 2) and a smaller band of immatureCFTR of148 kDa in native rat tissues but not in IgG

Fig. 2. 8-Bromo-cAMP (8-BrcAMP)-de-pendent redistribution of CFTR to theapical surface of crypt cells is inhibitedby vesicle traffic interruption. Underunstimulated conditions confocal im-

ages of labeled sections reveal thatCFTR is distributed in a wide bandextending beneath the apical surface(A, arrows; as shown in Fig. 1D) and isredistributed to the apical surface after8-BrcAMP treatment (B, small arrows).Treatment with 8-BrcAMP and prima-quine results in an accumulation ofCFTR in the subapical compartment(C, arrows). D: control section labeledin the presence of preimmune serumreveals no label in the crypts. L, lu-men. Bar, 10 m.

Table 1. CFTR fluorescence values

Apical Fl Subapical Fl Ratio A/SA FlFluid,

g min1 cm1

PBS 190.1959.41 98.9336.56 2.141.03 0.290.048-BrcAMP 210.7238.93 43.3323.33 6.153.08 0.1050.03

P0.001* P0.005Nocodazole 8-BrcAMP 166.0570.03 131.5369.78 1.410.65 0.070.01

P0.001 P0.058-BrcAMP primaquine 125.5889.42 159.5471.95 0.970.92 0.040.01

P0.001 P0.005

Values for fluorescence (Fl) are expressed in pixels as means SD determined from a minimum of 30 crypts and 10 random sections. n 4 animals per condition. A, apical; S, subapical; 8-BrcAMP, 8-bromo-CAMP; CFTR, cystic fibrosis transmembrane conductance regulator. P

values: * vs. PBS, vs. 8-BrcAMP (t-test).



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controls (Fig. 3A, lane 1) as shown previously (1, 9).These results confirmed the success of the immunopre-cipitation.

Before surface biotinylation experiments, we con-firmed that NHS-biotin was effective in surface bioti-

nylation of freshly isolated crypt and villus enterocytesby immunofluorescence labeling with Texas redstreptavidin (not shown). Having confirmed that wecould detect CFTR in immunoprecipitates from iso-lated enterocytes, we then proceeded with surface bi-otinylation/immunoprecipitation studies on VIP-stim-ulated and control cells. Streptavidin detection ofR3194 or IgG immunoprecipitates (Fig. 3B) after bioti-nylation of VIP-stimulated (Fig. 3B, lane 4) and control(Fig. 3B, lane 5) cells revealed a band consistent withmature CFTR of175183 kDa (Fig. 3B) that wasmore intense in VIP-treated samples than control. The

total material from stimulated and control sampleswas carefully normalized for total protein, and theamounts of total immunoprecipitated CFTR were al-most identical as determined in parallel immunoblots.Densitometric analysis of the CFTR band in indepen-

dent experiments revealed a 3.8 1.7-fold (P 0.005)increase in surface biotinylated CFTR in immunopre-cipitates after VIP treatment (Fig. 3B). In addition toCFTR, we identified at least two other CFTR antibody-specific biotinylated bands in both VIP and controls(Fig. 3B), apparent molecular mass of162 and 110kDa, that appeared more prominent in VIP-stimulatedimmunoprecipitates. Although one of these bands ap-pears at a level that may be confused with immatureCFTR, analysis of that band revealed it to be a polypep-tide of162 kDa, 14,000 kDa larger than the size of

Fig. 3. A: detection of CFTR in immunoprecipitates from isolated rat intestinal cells. Western blot analysis ofCFTR immunoprecipitates with the antibody R3194 (lane 2) reveals a broad band of 170185 kDa consistent withmature glycosylated CFTR (arrow) and a smaller band of 148 kDa consistent with immature CFTR (band B),neither of which are detected in IgG immunoprecipitates (lane 1). B: cAMP-dependent exocytosis of CFTR bysurface biotinylation. Rat jejunal enterocytes were isolated from animals infused with either vehicle or VIP,biotinylated, and immunoprecipitated with anti-CFTR antibody R3194 or IgG. Streptavidin detection on blotsrevealed a protein band of 180 kDa (arrow) consistent with mature CFTR and 2 other bands (*) in cellsimmunoprecipitated from VIP (lane 4) and in vehicle-treated animals with R3194 (lane 5) but not in nonimmuneIgG immunoprecipitates (lane 3). Although the top * band appears similar to band B of CFTR, it has a largermolecular mass of162 kDa. Both antibody-specific bands appeared more prominent in VIP-treated immunopre-cipitates (lane 4) than in controls (lane 5). Graph shows that densitometric analysis of biotinylated CFTR bandfrom VIP immunprecipitates (below lane 4) revealed an almost 4-fold increase over control (below lane 5). Data aremeans SD from 4 independent experiments; the difference between the bars was statistically significant (P 0.005). C: surface levels of biotinylated lactase do not change after cAMP stimulation. Streptavidin detection ofimmunoprecipitates with an antibody to lactase (YBB2/61) after biotinylation reveals a single band of150 kDa

in cells from VIP (lane 8) and vehicle-treated (lane 9) rats consistent with lactase. No bands were identified in IgGimmunoprecipitates from either VIP (lane 6)- or vehicle (lane 7)-treated rats. D: normal rat jejunal enterocyteswere immunoprecipitated with R3194 (lane 11) or IgG (lane 10) and immunoblotted with an antibody to AKAP149.

A single band of149 kDa was detected in R3194 (lane 11) but not in IgG immunoprecipitates (lane 10).Experiments shown in each panel were repeated at least 4 times. E: enterocytes from rat jejunum remain polarizedafter isolation. Immunofluorescence label for lactase with the antibody YBB2/61 and detected with FITC goatanti-mouse secondary antibody reveals apical labeling for lactase on the brush border of isolated enterocytes. F:control enterocytes labeled in the absence of primary antibody. Bar, 30 m.



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immature band B of CFTR in the same preparation(Fig. 3A).

We explored the possibility that the results describedabove are due to leaking of sulfo-NHS-biotin into thecells and therefore labeling intracellular proteins in-cluding CFTR. We tested this hypothesis by perform-ing biotinylation/immunoprecipitation of CFTR in thepresence and absence of saponin. Streptavidin detec-

tion of R3194 immunoprecipitates after saponin treat-ment and biotinylation revealed at least one additionalband that could not be identified in non-saponin-treated immunoprecipiates. These experiments sug-gest that in our system of isolated enterocytes, theplasma membrane remains intact both in the presenceand absence of cAMP stimulation, preventing sulfo-NHS-biotin from entering the cytoplasm unless thecells are permeabilized with saponin.

We also entertained the possibility that cAMP maybe inducing a generalized exocytosis of membrane pro-teins. To test the specificity of CFTR exocytosis, weused the same biotinylation/immunoprecipitation pro-cedure to analyze the apical membrane protein lactase

in VIP-stimulated and control immunoprecipitateswith the antibody YBB2/61 as shown in immunofluo-rescence localization (Fig. 1, A and B). Streptavidindetection of biotinylated immunoprecipitates of lactasewith the antibody YBB2/61 or IgG is shown in Fig. 3C.Under the same conditions that we used to detectCFTR, blot analysis of biotinylated lactase immuno-precipitates revealed a single antibody-specific proteinband of150 kDa consistent with lactase in VIP andunstimulated controls (Fig. 3, lanes 8 and 9) but not inIgG immunoprecipitates of VIP-stimulated or control(Fig. 3, lanes 6 and 7; Ref. 29). Densitometric analysisrevealed no difference in the intensities of the biotin-

ylated lactase band identified in VIP or control immu-noprecipitates. These observations are consistent withour immunofluorescence data indicating no change inthe distribution of lactase in intestinal cells on VIPstimulation, and they suggest that cAMP stimulatesexocytosis of a specific population of apical membraneproteins that comprises CFTR but not lactase. In ad-dition, this result further supports the notion that thetwo additional bands in CFTR immunoprecipitateswere true antibody-specific immunoprecipitating pep-tides and not just contaminants.

In fact, we were puzzled by these two additionalbiotinylated bands that coimmunoprecipitated withCFTR and not with lactase under the same conditions.

One possible explanation for this observation is thatCFTR exists in a multiprotein complex that includesother membrane proteins, as suggested by others (33,35). CFTR was recently shown to be physically linkedto regulatory complexes containing PKA and PKA an-choring proteins (AKAPs), sodium-hydrogen exchangerregulatory factor (NHERF), and ezrin in a complexinsoluble scaffold (35). To test whether our immuno-precipitation conditions preserve some of the protein-protein interactions in that scaffold, we analyzedwhether AKAP coimmunoprecipitates with CFTR inour system. Western blot analysis was performed using

an antibody to AKAP 149, a PKA type II anchoringprotein that is highly expressed in the small intestine.The antibody recognized a specific protein band of 149kDa in CFTR immunoprecipitates (Fig. 3D, lane 11)but not in IgG controls (Fig. 3D, lane 10), indicatingthat we were indeed immunoprecipitating a multipro-tein complex.

DISCUSSION

In the current study, two independent techniqueswere used to confirm that physiological cAMP stimu-lation and vesicle traffic regulate the number of CFTRchannels on the surface of the rat small intestinalepithelium. This observation resolves the current con-troversy regarding the role of membrane traffic inregulating CFTR in the intestine. Although cAMP-dependent exocytosis of CFTR to the apical membranehas been demonstrated in villus CHE cells (4), thephysiological relevance of that observation remainsunknown. Our observation in this work that CFTR isregulated in vivo by cAMP-dependent vesicle traffic

and channel insertion in both crypt and villus cells inassociation with fluid secretion, however, providesstrong support for physiological membrane traffic reg-ulation of CFTR and intestinal anion secretion.

Both receptor (VIP)- and non-receptor (8-BrcAMP)-mediated cAMP agonists induced a redistribution ofCFTR from the subapical to the apical domain in the jejunum. The lack of change in the distribution oflactase in crypt cells after cAMP agonist stimulationconfirmed that the cAMP-dependent redistribution ofCFTR was specific, because lactase is not regulated bycAMP-dependent vesicle insertion (23, 26). To deter-mine whether the cAMP-induced redistribution ofCFTR from the subapical to the apical domain is reg-ulated by vesicle traffic, we disrupted vesicle traffic in vivo with primaquine and nocodazole. In rat jejunum,primaquine (0.1 mM) was a potent inhibitor of thecAMP-dependent shift of CFTR from the subapical tothe apical domain in the crypt and reduced the fluidsecretory response of 8-BrcAMP (Table 1). The accu-mulation of CFTR in the subapical compartment in thecrypt in the presence of primaquine is consistent withthe observations by others of its effect in inhibitingreceptor recycling and in producing an intracellularaccumulation of endocytosed receptors, blocking theexit of receptors from the early endosomes and recy-cling to the plasma membrane (41). The reduction in

the fluid secretory response to 8-BrcAMP in vivo in thepresence of primaquine (35%) supports the notionthat insertion of new CFTR channels into the mem-brane contributes importantly to augmenting fluid se-cretion.

These experiments, however, may allow an alterna-tive albeit nonexclusive interpretation. If CFTR is con-tinuously recycling between the apical domain and thesubapical compartment, primaquine may interrupt thecycle and accumulate CFTR in the early endosomalcompartment. The fluorescence measurements in con-focal images actually point to this scenario. In that



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case, we could conclude that the balance between exo-cytosis and endocytosis of CFTR is almost as importantas channel gating as a regulatory factor. Our observa-tions in the intestine support the results of recentstudies in oocytes demonstrating that primaquinedrastically reduced cAMP-dependent CFTR chloridecurrents and effectively blocked vesicle and proteintraffic (40). Further studies will be necessary to assess

the relative contributions of exocytosis and endocytosisto the steady-state levels of surface CFTR on cAMPstimulation.

Although the effects of microtubule disruption onCFTR distribution and fluid movement were less strik-ing than those of primaquine, they also suggest a roleof membrane traffic in regulating the number of CFTRchannels on the apical surface and provide support forthe previous observation that cAMP-dependent chlo-ride secretion in rat intestine is regulated by microtu-bules (14).

Surface biotinylation, a well-established techniqueused to assess the delivery of proteins to the plasma

membrane (30, 31), confirmed cAMP-stimulated CFTRexocytosis. However, the finding that other unidenti-fied polypeptides were coimmunoprecipitating withCFTR and were also upregulated by cAMP, as shownin Fig. 3, was unexpected. Our first interpretation wasthat other proteins possessing at least one ectoplasmicdomain capable of biotinylation may be nonspecificcontaminants of the immunoprecipitation. This, how-ever, was unlikely for the following reasons: 1) theseother biotinylated proteins were CFTR antibody spe-cific in the immunoprecipitation and did not appear incontrols immunoprecipitated with nonimmune IgG(Fig. 3B, lane 3); 2) the conditions for immunoprecipi-tation were rather stringent, detergents were present

in all washes, and one of the washes was performed inhigh salt (0.6 M KCl) conditions; and 3) the sucrase-isomaltase immunoprecipitation experiments (Fig. 3C)supported the notion that our immunoprecipitationswere clean (in those cases no additional bands wereobserved).

Another potential artifact that could explain biotiny-lation of multiple bands is damage to the plasma mem-brane during the isolation of enterocytes before bioti-nylation. This possibility was ruled out by verifyingTrypan blue exclusion in parallel cell suspensions andby actually permeabilizing some cell suspensions withsaponin. The latter resulted in an increase in the

number of biotinylated bands, indicating that in theabsence of saponin the plasma membrane was tight.The observation that AKAP coimmunoprecipitateswith CFTR (Fig. 3D) in the intestine supports thenotion that the physiological regulation of CFTR byPKA involves a physical and functional associationwith AKAP as demonstrated recently (19). The coim-munoprecipitation of AKAP with CFTR indicated thatunder the conditions of homogenization, detergent sol-ubilization, and immunoprecipitation used here, theNHERF-ezrin insoluble scaffold that normally holdsCFTR (35, 36) is at least partially preserved.

At least one other transmembrane protein, Na/H

exchanger (NHE), is known to be attached to thisscaffold in addition to CFTR (42). Although the appar-ent molecular masses of the biotinylated bands that wefound do not correspond to that of NHE-3 (97 kDa; Ref.5), it is conceivable that other membrane proteins arealso attached to the same scaffold. Furthermore, be-cause some membrane proteins do not biotinylate and

because we cannot assert that the scaffold is totallyintact, the actual number of membrane proteins at-tached to the same scaffold of CFTR may be actuallygreater than three (CFTR and the 2 unknown proteinsfound in this work). On the other hand, the data pre-sented here do not rule out the possibility that theadditional unidentified proteins may be directly boundto CFTR and not to a NHERF-type scaffold. Identifica-tion of these proteins in future investigations will beimportant before any mechanistic model can be postu-lated.

In previous work from our laboratory (7) and others(27), it was found that cAMP stimulates exocytosis ofapical membrane proteins at a post-Golgi step. Thelack of effect of cAMP stimulation on lactase wouldsuggest, however, that cAMP-dependent exocytosis isrestricted to a subpopulation of apical membrane pro-teins. It has been generally accepted that cAMP oper-ates by increasing membrane traffic (7, 27). If that isthe case, the results in this work would suggest that atleast two subpopulations of subapical vesicles mustexist, one that carries CFTR and other proteins regu-lated by cAMP-dependent delivery and another cAMP-independent pathway that facilitates the transport ofproteins such as lactase. Such a senario raises inter-esting questions regarding potentially different path-ways that may regulate the formation and sorting of

these two different subpopulations of vesicles. Another alternative explanation that by no meansexcludes differences in vesicle traffic pathways is thatcAMP may actually increase the number of bindingsites available in the scaffold itself. Because the scaf-fold contains PKA and AKAP, it is conceivable that itsbinding capacity may be modulated by cAMP. In thatscenario, cAMP stimulation would increase the num-ber of surface molecules for all the membrane proteinsthat bind to the same scaffold, disregarding the vesi-cles that transport them to the cell surface. In otherwords, retention in the apical domain would be respon-sible for the increase of surface CFTR and some otherproteins. In both cases, the results of this study suggest

that the increase in the number of CFTR channels onthe surface of intestinal epithelial cells on cAMP stim-ulation contributes substantially to regulating fluidsecretion and is regulated by vesicle traffic. The observations in this study should provide the basis for acritical examination of membrane traffic in the patho-genesis of CFTR-mediated diseases in the intestine.

We thank Dr. A. Quaroni for the generous gift of antibodies andDr. G. McLaughlin and M. Hernandez for technical assistance.

Present address of N. A. Ameen: Pediatric Gastroenterology andCell Biology, University of Pittsburgh School of Medicine, Pitts-burgh, PA 15213.



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